gy 555 (2007) 129–138www.elsevier.com/locate/ejphar

European Journal of Pharmacolo

Antinociceptive properties of diphenyl diselenide: Evidencesfor the mechanism of action

Lucielli Savegnago, Larissa G. Pinto, Cristiano R. Jesse, Diego Alves,Joao B.T. Rocha, Cristina W. Nogueira, Gilson Zeni ⁎

Laboratório de Síntese, Reatividade e Avaliação Farmacológica e Toxicológica de Organocalcogênios, Centro de Ciências Naturais e Exatas,Universidade Federal de Santa Maria, Santa Maria, CEP 97105-900, RS, Brazil

Received 15 March 2006; received in revised form 9 October 2006; accepted 9 October 2006Available online 17 October 2006

Abstract

The present study examined the acute toxicity and antinociceptive effects of diphenyl diselenide (PhSe)2, given orally (p.o.), in chemical andthermical models of pain in mice. Diphenyl diselenide (7.8–312 mg/kg, p.o.) did not cause mortality. This compound did not change plasma AST(aspartate aminotransferase) and ALT (alanine aminotransferase) activities as well as urea and creatinine levels in mice after 72 h of exposure.Diphenyl diselenide (1–100 mg/kg, p.o.) inhibited acetic acid-, capsaicin-, glutamate-, bradykinin(BK)- and phorbol myristate acetate (PMA)-induced pain. Diphenyl diselenide also reduced glutamate-, bradykinin-, PMA-induced paw oedema formation. Moreover, diphenyl diselenidecaused a significant increase in tail-immersion response latency time. Diphenyl diselenide co-injected subplantarly in association with glutamate-induced a significant reduction of the licking and in the paw oedema formation induced by glutamate. The local pre-treatment of mice with L-arginine, intraplantarly, restored antinociception caused by diphenyl diselenide or NG-nitro-L-arginine methyl ester (L-NAME) when analyzedagainst glutamate-induced nociception. The pre-treatment of mice with dithiothreitol (DTT) intraplantarly restored local antinociception caused bydiphenyl diselenide or 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) when analyzed against glutamate-induced nociception. These results indicatethat diphenyl diselenide produced antinociception in several models of pain through mechanisms that involve an interaction with not only nitrergicsystem but also via interaction with redox modulatory sites of glutamate receptors.© 2006 Elsevier B.V. All rights reserved.

Keywords: Diphenyl diselenide; Selenium; Antinociception; Glutamate; Nitric oxide system

1. Introduction

Selenium is largely known to develop its biological activityas an integral part of functional selenoproteins, includingglutathione peroxidase and thioredoxin reductase, whichcatalyze reactions essential to the protection of cellularcomponents against oxidative and free radical damage (Rotrucket al., 1973; Burk and Lane, 1983; Bock et al., 1991).

The interest in organoselenium biochemistry and pharmacol-ogy has increased in the last two decades due to a variety oforganoselenium compounds that possess biological activity(Nogueira et al., 2004). Accordingly, a number of novel

⁎ Corresponding author. Tel.: +55 55 32208140; fax: +55 55 32208978.E-mail address: gzeni@quimica.ufsm.br (G. Zeni).

0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2006.10.003

pharmaceutical agents which are selenium-based or which targetspecific aspects of selenium metabolism are under development(May, 1999; Nogueira et al., 2003;Meotti et al., 2004). In additionto their antioxidant property (Muller et al., 1984; Meotti et al.,2004), selenium compounds were found to have neuro-protective(Porciúncula et al., 2001; Rossato et al., 2002), antihypertensive,anticancer, antiviral, immunosuppressive, antimicrobial andantiinflammatory properties (Sies, 1993; Schewe, 1995; May,1999; Nogueira et al., 2003; Zasso et al., 2005).

Ebselen (an organoselenium compound) is a classicalantioxidant and well known as the most important glutathioneperoxidase mimetic agent (Muller et al., 1984; Daiber et al.,2000). Furthermore to its peroxidase-like activity, ebselen hasantiinflammatory activity in different models of inflammation(Parnham and Graf, 1987; Schewe, 1995), which may be relatedat least in part to its capability to scavenge peroxynitrite, a

130 L. Savegnago et al. / European Journal of Pharmacology 555 (2007) 129–138

potent inflammatory mediator (Sies and Arteel, 2000). Themechanism(s) underlying the antiinflammatory activity ofebselen is still not completely understood but is linked toinhibition of NADPH-oxidase, protein kinase C, nitric oxidesynthase and lipoxygenases most likely by interacting withcritical thiol/disulfide groups in these enzymes (Cotgreave et al.,1989; Walther et al., 1999; Mugesh et al., 2001).

Recently, our group has reported that diphenyl diselenide, asimple diaryl diselenide, is active as a glutathione peroxidasemimic and is a safety drug when administered acutely to mice atdoses that have antiinflammatory and antinociceptive activities(Nogueira et al., 2003; Meotti et al., 2004). Moreover, data fromour laboratory have shown that diphenyl diselenide injectedsubcutaneously elicits significant and dose-related antinocicep-tion when assessed against chemical and thermal behaviouraltests. Moreover, our research group demonstrated that themechanism underlying the antinociception action of diphenyldiselenide involves the serotoninergic pathway (Zasso et al.,2005). Interestingly, the antinociceptive and antiinflammatorypotency of diphenyl diselenide was higher than that of ebselen(Nogueira et al., 2003, 2004).

Therefore, due to the need for more effective analgesic orantiinflammatory compounds with low side-effects, manystudies have been appearing in the literature involving thesynthesis of new compounds which can provide alternatives tocurrent therapeutic agents.

Based on the considerations above, the aims of the presentstudy were: (a) to evaluate the acute toxicity induced bydiphenyl diselenide in mice using oral route of administrationwith the purpose of offering safety in the use of this compound,(b) to investigate the antinociceptive activity induced bydiphenyl diselenide in chemical and thermal models of pain inmice and (c) to examine some possible mechanisms involved inthe local antinociceptive activity produced by diphenyldiselenide in the glutamate model of nociception.

2. Materials and methods

2.1. Drugs

Diphenyl diselenide (PhSe)2 was synthesized and character-ized in our laboratory by the method previously described(Paulmier, 1986). Analysis of the 1H NMR and 13C NMRspectra showed analytical and spectroscopic data in fullagreement with its assigned structure. The chemical purity ofdiphenyl diselenide (99.9%) was determined by GC/HPLC. Allother chemicals were of analytical grade and obtained fromstandard commercial suppliers. Diphenyl diselenide wasdissolved in DMSO (dimethyl sulfoxide, 10%) for theexperiments in which the local effect of (PhSe)2 was analyzed.The dose of DMSO (10%) did not cause detectable effect. Forthe other experiments, the vehicle used was canola oil.

2.2. Animals

The behavioural experiments were conducted using Swissmice of both sexes (25–35 g) maintained at 22–25 °C with free

access to water and food, under a 12:12 h light/dark cycle. In allexperiments of nociceptive behavioral, the animals (male andfemale mice were homogeneously distributed among groups)were acclimatized to the laboratory for at least 1 h before testingand were used only once throughout the experiments. Theexperiments of lethal-dose-response and toxicity of (PhSe)2were carried out only with male mice.

The animals were used according to the guidelines of theCommittee on Care and Use of Experimental Animal Resources,the Federal University of Santa Maria, Brazil and the ethicalguidelines for investigations of experimental pain in consciousanimals (Zimmermann, 1983). The number of animals andintensities of noxius stimuli used were minimum necessary todemonstrate the consistent effects of the drug treatments.

2.3. General toxicity and lethal-dose

To investigate the potential toxicity of diphenyl diselenide,mice received a single oral dose of diphenyl diselenide (7.8–312mg/kg) or vehicle (10 ml/kg, canola oil). After the compoundadministration, animals were observed up to 72 h to determine thelethal potential of diphenyl diselenide. The body weight gain ofanimals was also recorded as a sign of general toxicity.

After 72 h of exposure, mice were slightly anesthetized forblood collection by heart puncture in tubes containing heparin.Plasma was obtained by centrifugation at 2000 ×g for 10 min(hemolyzed plasma was discarded) and used for biochemicalassays.

The following biochemical parameters were measured:plasma enzymes AST (aspartate aminotransferase) and ALT(alanine aminotransferase) used as the biochemical markers forthe early acute hepatic damage and determined by thecolorimetric method of Reitman and Frankel (1957). Renalfunction was analyzed by determining plasma urea (Mackayand Mackay, 1927) and creatinine levels (Jaffe, 1886).

2.4. Acetic acid-induced abdominal constriction

The abdominal constrictions were induced by intraperitonealinjection of acetic acid (1.6%, i.p.) and carried out according tothe procedure described previously (Nogueira et al., 2003).After acetic acid injection, mice were individually placed inseparate boxes and the abdominal constrictions were countedcumulatively over a period of 20 min. Mice were pre-treatedwith diphenyl diselenide (1–100 mg/kg) by oral route (p.o.),30 min before the irritant injection. Control animals received asimilar volume of vehicle (10 ml/kg, canola oil).

To assess the time-course of the antinociceptive effect ofdiphenyl diselenide, mice were pre-treated with diphenyldiselenide (40 mg/kg, p.o.; this dose used was based on themean of ID50 for acetic acid test) 0.5–6 h before intraperitonealinjection of acetic acid.

2.5. Capsaicin-induced nociception

The method used for capsaicin-induced licking was similarto that described by Sakurada et al. (1993). After an adaptation

131L. Savegnago et al. / European Journal of Pharmacology 555 (2007) 129–138

period, mice were pre-treated with vehicle (canola oil, p.o.,10 ml/kg) or diphenyl diselenide (1–100 mg/kg, p.o.) 30 minbefore the intraplantar (i.pl.) administration of capsaicin(1.6 μg/paw, 20 μl) in the ventral surface of the right hindpaw.The amount of time spent licking the injected paw was recordedwith a chronometer for 5 min following capsaicin injection andwas considered as indicative of nociception.

2.6. Tail-immersion-induced nociception

The tail-immersion test was carried out as described byJanssen et al. (1963). The lower 3.5 cm portion of the tail wasmarked and animals were then injected with diphenyl diselenide(1–100 mg/kg, p.o.) or vehicle (canola oil, p.o., 10 ml/kg),30 min before the test. The test was determined by immersingthe lower 3.5 cm of the tail into a cup freshly filled with waterfrom a large constant-temperature (55 °C) bath until the typicaltail withdrawal response was observed. A 7-s cut-off wasimposed in this measure.

Changes in tail-flick latency, Δt (s), was calculated for eachanimal according to the formula:Δt (s)=post-drug latency−pre-drug latency (Pinardi et al., 2003).

In the experiments designed to evaluate the possibleparticipation of the opioid system in the antinociceptive effectof diphenyl diselenide, mice were pre-treated with naloxone(1 mg/kg, s.c.) or saline (0.9% NaCl) 15 min beforeadministration of vehicle (canola oil), morphine (2.5 mg/kg, s.c.) or diphenyl diselenide (10–100 mg/kg, p.o.). The tail-immersion test was carried out 30 min after morphine anddiphenyl diselenide administration.

2.7. Glutamate-induced nociception and paw oedema

The procedure used was similar to that described previously(Beirith et al., 2002). Mice were treated with diphenyldiselenide (1–100 mg/kg, p.o.) or canola oil (10 ml/kg, p.o.)30 min before glutamate injection. A volume of 20 μl ofglutamate solution (10 μmol/paw prepared in saline) wasinjected intraplantarly (i.pl.) in the ventral surface of the righthindpaw. The mice were observed individually for 15 minfollowing glutamate injection and the amount of time spentlicking the injected paw was recorded with a chronometer andwas considered as indicative of nociception.

To determine if the antinociceptive effect was due to a localaction, animals received 20 μl of glutamate solution (10 μmol/paw) co-administered with diphenyl diselenide (20–200 nmol/paw) intraplantarly in the ventral surface of the right hindpaw.After injection the drugs, mice were observed for 15 min and theamount of time spent licking the injected paw was recorded witha chronometer and was considered as indicative of nociception.The doses of diphenyl diselenide were selected on the basis ofprevious studies carried out in our laboratory (Zasso et al.,2005).

In order to assess whether the antinociceptive activityproduced by diphenyl diselenide (given orally or intraplantarly)in glutamate-induced nociception was associated with thedevelopment of oedema formation. The paw oedema was

measured by comparing the difference between the weight ofthe glutamate-treated paw and the weight of the contralateralpaw (non-treated paw). For this purpose, animals weresacrificed 15 min after glutamate injection by cervicaldislocation and both paws were cut at the ankle joint andweighed on an analytical balance.

2.8. Analysis of the mechanisms involved in diphenyl diselenideaction on the glutamate test

To address some mechanisms involved in local effect causedby diphenyl diselenide on glutamate-induced nociception andpaw oedema, distinct groups of animals were treated withdifferent classes of drugs, all of them locally co-administeredwith glutamate (10 μmol/paw).

To explore the possible contribution of nitric oxide-L-arginine pathway in the antinociception caused by diphenyldiselenide in the glutamate test, mice were pre-treated with L-arginine (60 μg/paw, i.pl., a nitric oxide precursor) and after10 min received diphenyl diselenide (200 nmol/paw, i.pl.),NG-nitro-L-arginine methyl ester (L-NAME,10 μg/paw, i.pl.,an inhibitor of nitric oxide synthase-NOS) or vehicle (20 μl/paw, i.pl.), all drugs were administered in association withglutamate (10 μmol/paw, i.pl.), except L-arginine. Afterinjection of the drugs, the mice were observed for 15 minand the amount of time spent licking the injected paw wasrecorded with a chronometer and was considered as indicativeof nociception.

To examine the involvement of redox modulatory site ofglutamate receptors in nociceptive transmission, sulfhydrylreducing agents such as dithiothreitol (DTT) and oxidizingagents, such 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) weretested. For this purpose, mice were pre-treated with dithiothreitol(0.3 μg/paw, i.pl.) and after 10 min they received diphenyldiselenide (200 nmol/paw, i.pl.), 5,5′-dithio-bis-(2-nitrobenzoicacid) (0.4 μg/paw, i.pl.) or vehicle (20 μl/paw, i.pl.), all drugswere administered in association with glutamate (10 μmol/paw,i.pl.), except dithiothreitol. After injection of drugs, the micewere observed for 15 min and the amount of time spent lickingthe injected paw was recorded with a chronometer and wasconsidered as indicative of nociception.

At the end of each experiment, we measured the paw oedemaas described by Beirith et al. (2002).

2.9. Bradykinin-induced nociception and paw oedema

The experiment was performed according to the methoddescribed by Ferreira et al. (2003). Mice were treated withdiphenyl diselenide (1–100 mg/kg, p.o.) or with canola oil(10 ml/kg, p.o.) 30 min before bradykinin injection (10 nmol/paw, 20 μl) in the ventral surface of the right hindpaw. Animalswere observed individually for 10 min following bradykinininjection and the amount of time spent licking the injected pawwas recorded with a chronometer and was considered asindicative of nociception. The paw oedema also was measuredat the end of experiment. All procedures were realized asdescribed by Cunha et al. (2001).

Table 2Effect of diphenyl diselenide administered orally on biochemical parameters inmice

Groups AST ALT Urea Creatinine(mg/kg) (U/l) (U/l) (mg/dl) (mg/dl)

Control 173.2±40.6 32.1±14.4 42.8±11.8 0.20±0.097.8 150.3±53.6 29.3±10.5 38.5±5.2 0.28±0.1615.6 187.8±52.0 36.5±16.4 41.1±6.9 0.16±0.0523.4 230.3±66.0 35.5±11.6 35.0±7.5 0.18±0.0731.2 152.3±18.5 27.0±10.4 36.0±7.6 0.20±0.0646.8 117.0±19.0 28.0±13.0 38.3±14.0 0.16±0.0562.4 140.5±34.3 18.8±4.3 32.1±7.1 0.18±0.04109.2 218.3±83 24.3±13 32.3±8.6 0.20±0.06156 178±44 25.3±16.4 31.5±6.3 0.15±0.05234 181.8±43.4 25.5±12.3 31.6±5.0 0.15±0.05312 244±56.8 32.1±26.3 35.3±25.2 0.18±0.04

Data are reported as mean±S.D. of six animals.

132 L. Savegnago et al. / European Journal of Pharmacology 555 (2007) 129–138

2.10. Phorbol myristate acetate (PMA)-induced nociceptionand paw oedema

The procedure used was similar to that described previously(Siebel et al., 2004). Mice were treated with diphenyl diselenide(1–100 mg/kg) by p.o. route, 30 min before i.pl. injection ofPMA (protein kinase C (PKC) activator, 0.03 μg/paw, 20 μl).After 15 min the i.pl. injection of PMA, animals were observedfor a further 30 min. The time spent licking the injected pawduring this period was recorded with a chronometer andconsidered as indicative of pain.

We also measured the paw oedema 45 min after PMAinjection. The procedure was realized as described previously(Siebel et al., 2004).

3. Statistical analysis

The results are presented as means±S.E.M. or means±S.D.The statistically significant difference between groups wascalculated by means of one-way analysis of variance (ANOVA)followed by Duncan's test when appropriate. Probability valuesless than 0.05 (Pb0.05) were considered as statisticallysignificant. The ID50 values (i.e. the dose of diphenyl diselenidewhich reduced the pain response by 50% in relation to controlgroup values) and LD50 values (i.e. the dose of diphenyldiselenide that caused mortality in 50% of mice) weredetermined by linear regression from individual experimentsusing “GraphPad Software” (GraphPad software, San Diego,CA) and are reported as geometric means accompanied by theirrespective 95% confidence limits. Maximal inhibition valueswere calculated at the more effective dose used.

4. Results

4.1. General toxicity and lethal-dose determination

Calculated LD50 for diphenyl diselenide administered byoral route was estimated to be N312 mg/kg (or 1 mmol/kg) withan observation period of 72 h. No mortality was observed afterthe mice exposure to all doses tested. However, a significant

Table 1Effect of diphenyl diselenide, administered orally, on the body weight gain inmice

Groups (mg/kg) Body weight gain (g)

Control 0.75±0.687.8 −1.90±0.62a

15.6 −1.93±1.75a

23.4 −3.25±1.25a

31.2 −2.83±1.16a

46.8 −4.61±1.86a

62.4 −2.41±1.43a

109.2 −4.41±1.08a

156 −3.08±1.73a

234 −3.51±2.63a

312 −5.20±1.92a

Data are reported as mean±S.D. of 6–12 animals.a Significantly different from the control group (Pb0.05).

reduction in the body weight gain was observed after diphenyldiselenide exposure, at all doses, when compared to the controlgroup (Table 1).

Moreover, oral administration of diphenyl diselenide inmice, at all doses tested, did not change plasma AST and ALTactivities, as well as, urea and creatinine levels when comparedto the control group (Table 2).

4.2. Acetic acid-induced abdominal constriction

A time-course analysis of the antinociceptive profile ofdiphenyl diselenide was accomplished. The antinociceptiveeffect of diphenyl diselenide reached its peak 30 min after p.o.administration and remained significant up to 2 h after theadministrations (results not show). Thus, the time point(30 min) of the maximum effect of diphenyl diselenide waschosen for all further studies.

The results of antinociceptive effect of diphenyl diselenideon acetic acid-induced abdominal constriction response in miceare presented in Fig. 1. It can be seen that diphenyl diselenide,given (30 min earlier) by oral route, produced an inhibition ofthe acetic acid-induced abdominal constriction in mice with the

Fig. 1. Effect of diphenyl diselenide administered orally against acetic acid-induced writhing movements in mice. Animals were pre-treated orally withdiphenyl diselenide at various doses (from 1 to 100 mg/kg) for 30 min prior tothe acetic acid (1.6%, i.p.). Each column represents the mean with S.E.M. for 6–12 mice in each group. Control (C) indicates the animal injected with canola oil.Asterisks denote the significance levels, when compared to the control group(one-way ANOVA followed by Duncan's test) ⁎ Pb0.05.

Fig. 2. Effect of diphenyl diselenide administered orally against capsaicin-induced licking in mice. Animals were pre-treated orally diphenyl diselenidewith at various doses (from 1 to 100 mg/kg) for 30 min prior to capsaicin(1.6 μg/paw). Each column represents the mean with S.E.M. for 8–12 mice ineach group. Control (C) indicates the animal injected with canola oil. Asterisksdenote the significance levels, when compared to the control group (one-wayANOVA followed by Duncan's test) ⁎ Pb0.05.

Table 3Effect of diphenyl diselenide administered orally on the licking and oedemainduced by glutamate in mice

Experimentalgroups

Glutamate

Licking (s) Paw oedema (mg)

Control 122.6±4.39 72.0±2.34(PhSe)2 (mg/kg) 1 119.5±12.3 68.1±4.095 49.6±6.01a 62.6±2.4510 37.0±4.71a 59.1±3.69a

25 20.7±4.75a 60.5±2.71a

50 12.8±3.29a 56.0±4.04a

100 5.5±1.33a 52.1±2.27a

Animals were pre-treated with diphenyl diselenide, (PhSe)2, at various doses(from 1 to 100 mg/kg, p.o.) for 30 min prior to glutamate (10 μmol/paw, i.pl.,20 μl). Data represents the mean with S.E.M. for 6–10 mice in each group.Control values (C) indicate the animal injected with canola oil. a Significantlydifferent from the control group (Pb0.05), as determined by one-way ANOVAfollowed by Duncan's test.

133L. Savegnago et al. / European Journal of Pharmacology 555 (2007) 129–138

mean ID50 value (and their respective 95% confidence limits) of39.72 (34.81–45.32) mg/kg and inhibition of 71±5%.

4.3. Capsaicin-induced nociception

Diphenyl diselenide, at all doses, caused an inhibition of thecapsaicin-induced licking response (Pb0.05, Duncan's test)(Fig. 2), with a mean ID50 value of 22.5 (13.3–38.1) mg/kg anda maximal inhibition value of 67±4%.

4.4. Tail-immersion-induced nociception

In the tail-immersion test the one-way ANOVA revealed thatdiphenyl diselenide administered, p.o., at the doses of 10–100mg/kg caused a significant increase in the reaction time to thermalstimuli as compared to the control group (Pb0.05) (Fig. 3).

Fig. 3. Effect of diphenyl diselenide administered orally on the tail-immersiontest in mice. Animals were pre-treated orally with diphenyl diselenide at variousdoses (from 1 to 100 mg/kg) for 30 min prior to tail-immersion at 55 °C. Eachcolumn represents the mean with S.E.M. for 6–10 mice in each group. Control(C) indicates the animal injected with canola oil. Asterisks denote thesignificance levels, when compared to the control group (one-way ANOVAfollowed by Duncan's test) ⁎ Pb0.05.

Pre-treatment with naloxone (1 mg/kg, s.c.), an antagonist ofthe opioid receptor, abolished the antinociceptive effect ofmorphine (positive control) at the dose of 2.5 mg/kg, s.c. Theantinociceptive effect of diphenyl diselenide (10–100 mg/kg, p.o.) was not prevented by pre-treatment with naloxone (resultsnot shown).

4.5. Glutamate-induced nociception and paw oedema

The results presented in Table 3 show that diphenyldiselenide, given orally, caused a significant inhibition of theglutamate-induced nociception, with a mean ID50 value of 14.2(8.8–22.2) mg/kg and inhibition 95±1%. Furthermore, one-way ANOVA revealed that the p.o. treatment of the animalswith diphenyl diselenide (10–100 mg/kg) resulted in aninhibition of the paw oedema formation induced by i.pl.injection of glutamate (Table 3). The maximal inhibitionobserved was of 28±3%.

When co-injected subplantarly in association with glutamate(10 μmol/paw), diphenyl diselenide (200 nmol/paw) caused asignificant reduction of the licking induced by glutamate. Themaximal inhibition observed was 47±5% (results not shown).Diphenyl diselenide, at all doses tested, inhibited of the pawoedema formation induced by injection of glutamate. Themaximal inhibition observed was 20±5% (results not shown).

4.6. Analysis of the possible peripheral mechanisms involved indiphenyl diselenide action on the glutamate test

The result presented in Fig. 4A shows that the local pre-treatment of mice with the nitric oxide precursor, L-arginine(60 μg/paw, i.pl.), intraplantarly injected 10 min earlier, restoredthe local antinociception caused by diphenyl diselenide(200 nmol/paw) or NG-nitro-L-arginine methyl ester (L-NAME; 10 μg/paw), when analyzed against glutamate-inducednociception. The paw oedema induced by the injection ofglutamate was reduced by diphenyl diselenide (200 nmol/paw)(Fig. 4B).

Fig. 5. Effect of pre-treatment of animals with dithiothreitol (DTT; 0.3 μg/paw)on the antinociceptive profiles of diphenyl diselenide ((PhSe)2; 200 nmol/paw)and 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB; 0.4 μg/paw) against theglutamate (10 μmol/paw, 20 μl)-induced licking (A) and paw oedema (B) inmice. Each column represents the mean with S.E.M. for 6–12 mice in eachgroup. ⁎ Pb0.05 when compared to the glutamate group plus vehicle (one-wayANOVA followed by Duncan's test), # Pb0.05 comparing diphenyl diselenideor DTNB plus DTT plus glutamate versus glutamate plus diphenyl diselenide orDTNB.

Table 4Effect of diphenyl diselenide administered orally on the licking and paw oedemainduced by bradykinin in mice

Experimentalgroups

Bradykinin

Licking (s) Paw oedema (mg)

Control 37.7±2.25 61.5±3.31(PhSe)2 (mg/kg) 1 34.0±6.31 60.0±2.885 28.0±5.22 58.5±5.3310 27.0±6.04 57.2±5.3525 10.7±4.81a 57.7±4.7550 8.7±2.75a 59.7±4.32100 3.4±1.34a 46.5±3.30a

Animals were pre-treated with diphenyl diselenide, (PhSe)2, at various doses(from 1 to 100 mg/kg, p.o.) for 30 min prior to bradykinin (10 nmol/paw, i.pl.,20 μl). The paw oedema was measured at the end of experiment. Data arereported as the mean with S.E.M. for 8–10 mice in each group. Control values(C) indicate the animal injected with canola oil. a Significantly different from thecontrol group (Pb0.05), as determined by one-way ANOVA followed byDuncan's test.

Fig. 4. Effect of pre-treatment of animals with L-arginine (60 μg/paw) on theantinociceptive profiles of diphenyl diselenide ((PhSe)2; 200 nmol/paw) andNG-nitro-L-arginine methyl ester (L-NAME; 10 μg/paw) against the glutamate(10 μmol/paw, 20 μl)-induced licking (A) and paw oedema (B) in mice. Eachcolumn represents the mean with S.E.M. for 6–10 mice in each group. ⁎ Pb0.05when compared to the glutamate group plus vehicle (one-way ANOVA followedby Duncan's test), # Pb0.05 comparing agonist (diphenyl diselenide or L-NAME) plus antagonist (L-arginine) plus glutamate versus agonist plusglutamate.

134 L. Savegnago et al. / European Journal of Pharmacology 555 (2007) 129–138

The participation of redox modulatory site of glutamatereceptors in nociceptive transmission in glutamate-inducedlicking and paw oedema was confirmed by using dithiothreitoland 5,5′-dithio-bis-(2-nitrobenzoic acid). The pre-treatment ofmice with dithiothreitol, given 10 min earlier, restored theantinociception caused by diphenyl diselenide or 5,5′-dithio-bis-(2-nitrobenzoic acid) when analyzed against glutamate-induced nociception (Fig. 5). Conversely, the paw oedema wasnot altered by dithiothreitol (results not shown).

4.7. Bradykinin-induced nociception and paw oedema

As can be seen in Table 4, diphenyl diselenide (25, 50 and100 mg/kg, p.o.) significantly inhibited nociception induced byi.pl. injection of bradykinin (10 nmol/paw). The maximalinhibition observed was 91±4% and the calculated mean ID50

value for this effect was 34.66 (24.26–49.52) mg/kg.

Furthermore, diphenyl diselenide (100 mg/kg, p.o.) wassignificantly effective in inhibiting the mouse paw oedemainduced by i.pl. injection of bradykinin (percentage of inhibitionof 24±4%)(Table 4).

4.8. Phorbol myristate acetate (PMA)-induced nociception andpaw oedema

As revealed in Table 5, oral treatment with diphenyl diselenide(1–100 mg/kg) significantly inhibited PMA-induced lickingresponse. The maximal inhibition observed was 92±2% and thecalculated mean ID50 value for this effect was 31.09 (20.94–46.17) mg/kg. Oral treatment of the animals with diphenyl

Table 5Effect of diphenyl diselenide administered orally on the licking and paw oedemainduced by PMA in mice

Experimentalgroups

PMA

Licking (s) Paw oedema (mg)

Control 141.1±9.96 49.85±3.21(PhSe)2 (mg/kg) 1 137.7±8.43 42.4±3.2710 41.7±4.92a 43.5±3.3050 30.4±4.55a 42.4±1.10100 11.5±2.50a 33.2±1.93a

Animals were pre-treated with diphenyl diselenide, (PhSe)2, at various doses(from 1 to 100 mg/kg, p.o.) for 30 min prior to PMA (0.03 μg/paw, i.pl., 20 μl).The paw oedema was measured 45 min after PMA injection. Data are reportedas the mean with S.E.M. for 8–10 mice in each group. Control values (C)indicate the animal injected with canola oil. a Significantly different from thecontrol group (Pb0.05), as determined by one-way ANOVA followed byDuncan's test.

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diselenide resulted in an inhibition of 34±4% of the paw oedemaformation induced by i.pl. injection of PMA (Table 5).

5. Discussion

In the present study, we found that diphenyl diselenideadministered orally produced minor toxicological effects andinduced antinociception in various pain models in mice. Theminor toxic potential of diphenyl diselenide, orally adminis-tered, was evidenced by the index of mortality. Althoughdiphenyl diselenide did not provide evidences for renal orhepatic toxicity, the body weight gain in mice exposed to thiscompound was reduced, which might indicate systemic toxicity.

Previous report demonstrated that the toxicity induced bydiphenyl diselenide depends on the route of administration aswell as the species (rat or mice) (Nogueira et al., 2003). In fact,diphenyl diselenide administered by i.p. route was more toxicthan by s.c. route in mice (LD50=62.4 and N156 mg/kg,respectively) (Nogueira et al., 2003). Here, we demonstrated thatLD50 for diphenyl diselenide administrated orally was similar tothat found by s.c. route. Accordingly, diphenyl diselenideadministered by s.c. or p.o route did not induce seizures or deathin mice, which can be attributed to a slow rate of absorption andlack of metabolism of the compound. In contrast, this compoundinjected by i.p. route induced seizures and death in mice, whichcan be tentatively linked to a faster rate of absorption and to afirst pass liver metabolism (Nogueira et al., 2003).

The results presented in this study revealed that diphenyldiselenide, orally administered, elicited an inhibition of theacetic acid-induced visceral nociceptive response in mice.Accordingly, this organoselenium compound reduced visceralpain induced by acetic acid when subcutaneously administered(Nogueira et al., 2003). The acetic acid-induced writhingreaction in mice, described as a model for visceral pain, has longbeen used as a screening tool for the assessment ofantinociceptive or antiinflammatory property of new agents(Vinegar et al., 1979; Tjϕsen and Hole, 1997).

One interesting finding in the present study was theobservation that diphenyl diselenide orally injected caused aprolonged tail-flick latency, indicating an increase of nocicep-

tive threshold. The results obtained on tail-flick test confirmedthat antinociception caused by diphenyl diselenide was notmediated by opioid mechanisms. These data are in accordancewith previous data demonstrating that diphenyl diselenidesubcutaneously administered caused dose-related increase inthe latency for tail-flick response, similar to morphine(Nogueira et al., 2003). The tail-flick response is believed tobe a spinally mediated reflex (Chapman et al., 1985). Moreover,Grumbach (1966) has shown that the effectiveness ofantinociceptive agents in the tail-flick pain model is highlycorrelated with relief of human pain.

We have also shown, in the present study, that diphenyldiselenide produced significant antinociceptive effect on thecapsaicin-induced neurogenic paw licking response. Theseresults are consistent with our previous work, in which, diphenyldiselenide administered by subcutaneous route caused signifi-cant antinociception against capsaicin-induced licking(Nogueira et al., 2003). It has been shown that capsaicin, thepungent algesic substance obtained from hot red chili peppers, isa valuable pharmacological tool for studying a subset ofmammalian primary sensory C-fibers and Aδ afferent neuronsincluding polymodal nociceptors and warm thermoceptors(Jancsó, 1992). In addition, it has been proposed thatcapsaicin-induced nociception is brought about by activationof the capsaicin receptor, also known as the vanilloid receptor(TRPV), termed TRPV subtype 1 (TRPV1), ligant-gated non-selective cation channel in primary sensory neurons (Caterinaet al., 1997; Tominaga et al., 1998; Szallasi and Blumberg,1999). It has been widely documented that a capsaicin evokesthe release of neuropeptideos, excitatory amino acids (glutamateand aspartate), nitric oxide and pro-inflammatory mediators inthe periphery and transmits nociceptive information to the spinalcord (Santos and Calixto, 1997; Sakurada et al., 1996, 2003).

Based on this evidence, we sought to determine whether ornot diphenyl diselenide inhibits nociceptive response caused byintraplantar injection of glutamate into the mouse hindpaw. Theresults demonstrated that diphenyl diselenide, administeredorally, produced a significant inhibition in the licking and thepaw oedema caused by intraplantar injection of glutamate intothe mouse hindpaw. It is interesting to mention that diphenyldiselenide was more potent to inhibit the nociception caused byglutamate (ID50=14.2 mg/kg) than that of caused by capsaicin,bradykinin, PMA and acetic acid (22.5, 34.6, 31.09 and39.7 mg/kg, respectively) in mice. In fact, diphenyl diselenidewas significantly more potent in blocking glutamate andcapsaicin-induced nociception. Thus, the present resultsindicate that, at least in part, the antinociceptive action causedby diphenyl diselenide could be due to an interaction with thecapsaicin receptor and the glutamatergic system. In agreement,diphenyl diselenide modulates glutamatergic system in vivo andin vitro (Nogueira et al., 2001).

Glutamate plays an important role in nociception peripheralpain transmission and its intraplantar injection evokes thermaland mechanical hyperalgesia (Jackson et al., 1995; Carltonet al., 1995, 1998) as well as spontaneous lifting and lickingbehaviors in mice (Beirith et al., 2002). Glutamate excites andsensitizes nociceptors localized in the unmyelinated axons of rat

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glabrous skin (Coggeshall and Carlton, 1998). It has beenreported the presence of peripheral glutamate receptors onperipheral primary afferents in humans (Warncke et al., 1997).

In order to provide more evidence of glutamatergic systeminvolvement in antinociception caused by diphenyl diselenide,we attempted to determine whether or not diphenyl diselenideco-injected locally with glutamate was capable of inhibitingnociceptive response caused by glutamate into the mousehindpaw. In agreement with the findings obtained, we revealed,for the first time, that local administration of diphenyl diselenideinduces antinociceptive and antioedematogenic effect when co-injected with glutamate. In agreement, Zasso et al. (2005) havereported that diphenyl diselenide produced a significantantinociceptive local effect on the late phase of the formalin test.

In the present study we also attempted to characterize furthersome of the mechanisms through which diphenyl diselenideexerts its local antinociceptive action in the glutamate model ofnociception in mice.

To our knowledge, this is the first study indicating that L-arginine-nitric oxide pathway is, at least in part, involved inantinociception induced by diphenyl diselenide in the glutamatetest. This conclusion derives from the fact that pre-treatment ofanimals with the substrate for NOS, L-arginine, significantlyrestored the antinociception caused by diphenyl diselenide andNG-nitro-L-arginine methyl ester (L-NAME; a known inhibitorof nitric oxide synthase). Considering the present data, we mayspeculate that local antinociceptive action produced by diphenyldiselenide is probably linked to an inhibition of NOS activity.Considerable evidence has accumulated suggesting a role fornitric oxide (NO) as a mediator of inflammation (Lyons, 1995).NO increases the synthesis/release of pro-inflammatory med-iators such as cytokines and reactive oxygen species (Marcin-kiewicz et al., 1995) and prostanoids (Sautebin et al., 1995),resulting in promotion of inflammatory reaction. In this way,peripherally released NO contributes to the development ofoedema and hyperalgesia in tissue injury and inflammation(Peana et al., 2006).

Another interesting finding in the present study is thedemonstration that the pre-treatment of mice with dithiothreitolsignificantly restored the antinociception caused by diphenyldiselenide and 5,5′-dithio-bis-(2-nitrobenzoic acid). Here, wereported that a reducing agent, dithiothreitol, caused nocicep-tion and 5,5′-dithio-bis-(2-nitrobenzoic acid), the oxidizingagent, induced significant alleviation of the nociceptiveresponse caused by injection of glutamate. Reynolds et al.(1990) found that reducing agents potentiate the NMDAreceptor-induced increase in intracellular calcium, and thiseffect has been shown to be reversed by 5,5′-dithio-bis-(2-nitrobenzoic acid). NMDA receptor function is highly regulatedby various modulating sites located on the receptor, includingthe redox modulatory (Rock and MacDonald, 1995; Laughlinet al., 1998). This result may indicate that the antinociceptiveeffect induced by diphenyl diselenide could be via theinteraction with redox modulatory site of glutamate receptors,namely NMDA receptor.

Recently, it has been established that nociceptive responseinduced by glutamate is mediated by interaction with NMDA

receptors and the activation of the L-arginine-nitric oxidepathway (Beirith et al., 2002). Thus, these previous findings andthe present results may suggest that the local antinociceptiveaction induced by diphenyl diselenide could be both aninhibition of NOS and a blockade of NMDA receptor.

The current study clearly indicates that the antinociceptiveeffect of diphenyl diselenide, given by p.o. route, is related tothe release of bradykinin. In fact, diphenyl diselenide inhibitedbradykinin-induced antinociception and paw oedema formationin mice. Bradykinin and its related kinins are vasoactivepeptides which have an important role as inflammatorymediators and are normally released following tissue traumaor infection. Once released, bradykinin may induce pain bydirect stimulation of the nociceptors (Aδ and C-fibers)innervating many tissues. Furthermore, bradykinin can releasemost inflammatory and algogenic substances, namely productsderived from arachidonic acid pathways, cytokines and nitricoxide (Dray and Perkins, 1997; Calixto et al., 2000, 2001).

In this study we have also demonstrated that diphenyldiselenide, orally injected, inhibited PMA-induced antinocicep-tion and paw oedema formation in mice. In fact, Souza et al.(2002) have reported that an intraplantar injection of PMA, aknown protein kinase C activator, induces paw licking in mice.Accordingly, Tsuchiya et al. (2005) found that nociceptiveresponse to PMAwas associated with the formation of oedemaand erythema in the injected paw which was observed inhistological study.

In conclusion, our results indicate that diphenyl diselenide isa drug which induced minor toxic effects when administered byp.o route in mice. Additionally, this compound may be a usefuldrug as an antiinflammatory and an antinociceptive agent sincediphenyl diselenide produced antinociception in differentmodels of pain in mice. Besides, the antinociceptive action ofthis compound could be associated with its ability to inhibit NOproduction and through the interaction with the glutamatergicsystem.

Acknowledgements

The financial support by UFSM (FIPE), FAPERGS, CAPESand CNPq are gratefully acknowledged. J.B.T.R., L.S., C.R.J.,C.W.N. and G.Z. are the recipients of CNPq fellowships.

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